Peptides used in the study and sequence homologies.

<p>Sequence homology between the AS peptide and self-proteins (colons indicate identity and asterisks indicate conservative substitutions).</p

Antibodies directed against the <i>Klebsiella pneumoniae-</i>derived DPP_121-145 peptide in the sera of patients with inflammatory arthritides.

<p>(A) The results of an assay of europium-labeled antihuman IgG antibodies are shown; each circle represents a measurement for one patient, and the dashed horizontal line indicates the cut-off value of 32,250 international units. ROC curves showing sensitivity and specificity of the assay of IgG antibodies against the <i>Klebsiella pneumoniae</i>-derived DPP_121-145 peptide for differentiating between AS sera and l healthy donors (B), between AS samples and RA (C) and between AS sera and PsA samples (D).</p

Anti- peptide antibodies bind <i>Klebsiella pneumoniae</i>-derived proteins.

<p>(A) Sequence homology between the AS peptide and the <i>Klebsiella pneumoniae</i>-derived proteins: sugar transporter (SET) protein, Xaa-Pro dipeptidase (DPP) protein and L-fucose isomerase (LFI) protein. Binding of affinity purified anti-AS peptide antibodies (black continuous line) to the SET (B) and LFI (C) peptide in DELFIA. Binding of affinity purified anti-AS peptide antibodies to recombinant DPP protein in ELISA (D). Dotted line: antibodies affinity purified against an irrelevant control peptide. X axis: increasing antibody concentration by two fold ranging from 1.25 microgram/ml to 10 microgram/ml. Y axis: IgG international units for DELFIA assay and Optical Density values obtained at 405 nm wavelength for ELISA assay.</p

Binding of anti-peptide antibodies to autoantigens.

<p>Binding of affinity purified anti-AS peptide antibodies to collagen type I (A), collagen type II (B), fibronectin (C), PER peptide (D), ADM peptide (E). Black continous line: antibodies affinity purified against the AS peptide from the sera of patients affected by AS. Dotted line: antibodies affinity purified against an irrelevant control peptide. A, B and C: ELISA assay. D and E: DELFIA assay. X axis: increasing antibody concentration by two fold ranging from 1.25 microgram/ml to 10 microgram/ml. Y axis: Optical Density values obtained at 405 nm wavelength for ELISA assay and IgG international units for DELFIA assay. F: western blot analysis of the binding of anti-peptide antibodies to ASAP1(molecular weight 125 KDa). Lane 1 and 2, antibodies affinity purified against an irrelevant control peptide probed with cell lysate from cells transfected with ASAP1 (lane 1) or from untransfected cells (lane 2); lane 3 and 4, antibodies affinity purified against the AS peptide probed with cell lysate from cells transfected with ASAP1 (lane 3) or from untransfected cells (lane 4); lanes 5 and 6, commercially available monoclonal antibody directed against ASAP1, probed with cell lysate from cells transfected with ASAP1 (lane 5) or from untransfected cells (lane 6). Lower panel of Fig 2F: immunoblot with actin demonstrates equal protein loading.</p

Image_1_CD34+DNAM-1brightCXCR4+ haemopoietic precursors circulate after chemotherapy, seed lung tissue and generate functional innate-like T cells and NK cells.jpeg

BackgroundThere is little information on the trajectory and developmental fate of Lin-CD34+DNAM-1bright CXCR4+ progenitors exiting bone marrow during systemic inflammation.ObjectiveTo study Lin-CD34+DNAM-1bright CXCR4+ cell circulation in cancer patients, to characterize their entry into involved lung tissue and to characterize their progenies.MethodsFlow cytometric analysis of PBMC from 18 patients with lung cancer on samples collected immediately before the first and the second treatment was performed to study Lin-CD34+DNAM-1bright CXCR4+ precursors. Precursors were purified (>99%) and cultured in vitro from all patients. Paired PBMC and tissue samples from patients undergoing tumor resection were analyzed by flow cytometry to assess tissue entry and compare phenotype and developmental potential of Lin-CD34+DNAM-1bright CXCR4+ cells in both compartments.ResultsSignificant circulation of Lin-CD34+DNAM-1bright CXCR4+ precursors was observed 20d after the first treatment. Precursors express CXC3CR1, CXCR3, CXCR1 consistent with travel towards inflamed tissues. Flowcytometric analysis of lung tissue samples showed precursor presence in all patients in tumor and neighboring uninvolved areas. Successful purification and in vitro culture from both blood and lung tissue generates a minor proportion of maturing NK cells (85%) of α/β T-progenies with innate-like phenotype expressing NKG2D,NKp30,DNAM-1. Innate-like maturing T-cells in vitro are cytotoxic, can be triggered via NKR/TCR co-stimulation and display broad spectrum Th1,Th2 and Th1/Th17 cytokine production.ConclusionIn advanced stage lung cancer CD34+DNAM-1brightCXCR4+ inflammatory precursors increase upon treatment, enter involved tissues, generate functional progenies and may thus represent an additional player contributing to immune balance in the highly SDF-1/CXCR4-biased pro-metastatic tumor microenvironment.</p

DataSheet_1_CD34+DNAM-1brightCXCR4+ haemopoietic precursors circulate after chemotherapy, seed lung tissue and generate functional innate-like T cells and NK cells.docx

BackgroundThere is little information on the trajectory and developmental fate of Lin-CD34+DNAM-1bright CXCR4+ progenitors exiting bone marrow during systemic inflammation.ObjectiveTo study Lin-CD34+DNAM-1bright CXCR4+ cell circulation in cancer patients, to characterize their entry into involved lung tissue and to characterize their progenies.MethodsFlow cytometric analysis of PBMC from 18 patients with lung cancer on samples collected immediately before the first and the second treatment was performed to study Lin-CD34+DNAM-1bright CXCR4+ precursors. Precursors were purified (>99%) and cultured in vitro from all patients. Paired PBMC and tissue samples from patients undergoing tumor resection were analyzed by flow cytometry to assess tissue entry and compare phenotype and developmental potential of Lin-CD34+DNAM-1bright CXCR4+ cells in both compartments.ResultsSignificant circulation of Lin-CD34+DNAM-1bright CXCR4+ precursors was observed 20d after the first treatment. Precursors express CXC3CR1, CXCR3, CXCR1 consistent with travel towards inflamed tissues. Flowcytometric analysis of lung tissue samples showed precursor presence in all patients in tumor and neighboring uninvolved areas. Successful purification and in vitro culture from both blood and lung tissue generates a minor proportion of maturing NK cells (85%) of α/β T-progenies with innate-like phenotype expressing NKG2D,NKp30,DNAM-1. Innate-like maturing T-cells in vitro are cytotoxic, can be triggered via NKR/TCR co-stimulation and display broad spectrum Th1,Th2 and Th1/Th17 cytokine production.ConclusionIn advanced stage lung cancer CD34+DNAM-1brightCXCR4+ inflammatory precursors increase upon treatment, enter involved tissues, generate functional progenies and may thus represent an additional player contributing to immune balance in the highly SDF-1/CXCR4-biased pro-metastatic tumor microenvironment.</p

Image_2_CD34+DNAM-1brightCXCR4+ haemopoietic precursors circulate after chemotherapy, seed lung tissue and generate functional innate-like T cells and NK cells.jpeg

BackgroundThere is little information on the trajectory and developmental fate of Lin-CD34+DNAM-1bright CXCR4+ progenitors exiting bone marrow during systemic inflammation.ObjectiveTo study Lin-CD34+DNAM-1bright CXCR4+ cell circulation in cancer patients, to characterize their entry into involved lung tissue and to characterize their progenies.MethodsFlow cytometric analysis of PBMC from 18 patients with lung cancer on samples collected immediately before the first and the second treatment was performed to study Lin-CD34+DNAM-1bright CXCR4+ precursors. Precursors were purified (>99%) and cultured in vitro from all patients. Paired PBMC and tissue samples from patients undergoing tumor resection were analyzed by flow cytometry to assess tissue entry and compare phenotype and developmental potential of Lin-CD34+DNAM-1bright CXCR4+ cells in both compartments.ResultsSignificant circulation of Lin-CD34+DNAM-1bright CXCR4+ precursors was observed 20d after the first treatment. Precursors express CXC3CR1, CXCR3, CXCR1 consistent with travel towards inflamed tissues. Flowcytometric analysis of lung tissue samples showed precursor presence in all patients in tumor and neighboring uninvolved areas. Successful purification and in vitro culture from both blood and lung tissue generates a minor proportion of maturing NK cells (85%) of α/β T-progenies with innate-like phenotype expressing NKG2D,NKp30,DNAM-1. Innate-like maturing T-cells in vitro are cytotoxic, can be triggered via NKR/TCR co-stimulation and display broad spectrum Th1,Th2 and Th1/Th17 cytokine production.ConclusionIn advanced stage lung cancer CD34+DNAM-1brightCXCR4+ inflammatory precursors increase upon treatment, enter involved tissues, generate functional progenies and may thus represent an additional player contributing to immune balance in the highly SDF-1/CXCR4-biased pro-metastatic tumor microenvironment.</p

Proliferative capacity of ALL-MSCs as compared with HD-MSCs.

<p>Calculated cumulative population doublings (PDs) from P1 to P5 of MSCs isolated from HDs and from ALL patients at diagnosis and at following time-points of treatment, namely day+15, +33 and +78. The data represent the mean of 10 HD-MSCs and 10 ALL-MSCs. P values less than 0.05 were considered to be statistically significant.</p

Mean percentage of proliferation of healthy donor peripheral blood mononuclear cells (PBMCs) stimulated with PHA in the presence of HD-MSCs, as compared with ALL-MSCs generated at day+0; +15; +33; +78 at two different MSC∶PBMC ratios (1∶2; 1∶10).

<p>Each value represents the mean ± SD of multiple experiments (each point being in triplicate). PBMC proliferation stimulated with PHA in the absence of MSCs was 71.80%. P values less than 0.05 were considered to be statistically significant.</p

<i>In vitro</i> life-span and senescence of ALL-MSCs as compared with HD-MSCs.

<p><i>In vitro</i> life-span of HD-MSCs (<b>A</b>) and ALL-MSCs at diagnosis (<b>B</b>), defined as number of passages before observation of senescence, from 10 representative donors and patients (#1–10). A large variability among HDs and patients was observed. (<b>C</b>): ß-Galactosidase staining of ALL-MSCs from patient #2 at passage (P) 18, isolated at day+0. Senescence of the cells is indicated by the positivity for the staining. <b>D and E:</b> cell cycle analysis performed by flow-cytometry on ALL-MSCs from patient #2 at early (P2) and late (P18) passages in culture. At P2 (D), the percentage of proliferating cells (in S phase) is 56.6%, whereas cells in G0/G1, G2 phase are 11.1 and 29.2%, respectively. The percentage of apoptotic cells in subG1 phase is 2%. At P18 (E), when cells become senescent, the percentage of proliferating cells decreases to 26.5%, whereas cells in G0/G1, G2 and subG1 phase are 57.5%, 2.6% and 12.3%, respectively.</p